The present invention relates to automated methods and apparatus for optically analyzing samples in biological sample containers such as well plates.
Biological samples such as animal cells, in particular mammalian cells, are commonly cultured in biological sample containers such as well plates (sometimes called microtiter plates or microplates), omni trays, Q-trays and Petri dishes. Much of the processing of the samples can be performed automatically using robotic apparatus that can deliver containers to and from various stations at which the samples can be observed and imaged using camera equipment, and transferred to other containers using an array of pins on a movable mechanical head.
FIGS. 1A and 1B schematically illustrate an example biological sample container 1 in the form of a well plate with a plurality of wells 5 in a 4×3 array. More typically a well plate used for automated processes on a robotic platform will have an array of wells, e.g. 6, 24, 96, 384, 1536, 3072 or 6144 etc wells, but may sometimes only have a single well. The spacing between the wells and/or the external dimensions of the plates relevant for handling, typically conform to the standard of the Society for Biomolecular Screening (SBS) adopted by the American National Standards Institute (ANSI) or derivates and extensions thereof used in the industry. The ANSI microplate standards include: SBS-1 2004: Footprint Dimensions; SBS-2 2004: Height Dimensions; SBS-3 2004: Bottom Outside Flange Dimensions; and SBS-4 2004: Well Positions of 96, 384, and 1536 well plates. The contents of these microplate standards are incorporated herein by reference, in particular the relevant dimensions.
For example, according to the SBS-4 standard, a 96 well plate has wells spaced apart in a square grid by 9 mm. The corresponding dimensions for 384 and 1536 well plates according to the SBS-4 standard are 4.5 mm and 2.25 mm respectively. Other well plates can have their well spacing dimension calculated on this basis, even if not explicitly covered by the SBS-4 standard, e.g. 24 well plates can be provided with an 18 mm inter-well spacing as an extrapolation of the ANSI standard.
Further, according to the SBS-1 standard, a well plate should have external dimensions of 127.76 mm (length)×85.48 mm (width)±certain specified tolerances.
References to standard dimensions in relation to well plates made in this document are thus made with reference to the above true standards defined by ANSI, and also derivates from and extensions of these standards used in the industry, as well as covering new standards that may be defined for well plates in the future.
Referring to FIG. 1A, the biological sample container 1 is a tray-like container having a top surface 2 with a number of wells 5 for accommodating biological or chemical samples. FIG. 1B is a schematic cross section along the line A-A′ of FIG. 1A. It can be seen from FIG. 1B that each of the wells 5 comprises a side wall 3 and a base 4. In the present case, the well plate 1 is one suitable for optical imaging so the base 4 of each well 5 is transparent, and optionally the side wall 3 also.
For successful imaging, it is necessary to be able to accurately position the sample in the field of view of an imaging camera, and to focus the camera on the plane of interest. For well plate imaging in automated processes, the focusing of the imaging camera needs to be carried out in an automated way, and autofocus systems are generally used in the art for this purpose.
U.S. Pat. No. 6,130,745[1] and U.S. Pat. No. 6,441,894[2] describe a prior art technique for focusing a laser beam used to excite fluorescence in cell colonies cultured in wells in a well plate. It is important to accurately position a tightly focused beam within the cell colony so as to avoid exciting fluorescence in unbound fluorescent markers outside the colony. The method involves focusing the laser beam near the lower surface of the base of a well, and detecting light reflected back. The basic principles of this method are schematically illustrated in FIGS. 2A to 2C. A well 15 contains a solution 18 which may contain sample cells to be imaged (not shown). FIG. 2A illustrates the arrangement at a first time t=t1, FIG. 2B illustrates the arrangement at a second time t=t2, and FIG. 2B illustrates the arrangement at a third time t=t3. In each case, the well 15 comprises a side wall 13 and a base 14. The base has a finite thickness as defined by an upper surface and a lower surface thereof. Referring first to FIG. 2A, a laser is disposed beneath the well 15, and light emitted from the laser is focused by a lens 16 to a focal point 17 near the lower surface of the base 14 of the well 15. Then, from the time t1 to the time t3, the focal point 17 is scanned upwards. The reflected light intensity reaches a maximum when the light is focused on the lower surface. This occurs at around the time t2 as shown in FIG. 2B. Thus, the lower surface of the well base is detected. The focus is then advanced upwards by at least the known thickness of the base so that the sample volume defined by the well is focused. It is noted that the base thickness is known from the specification of the well plate provided by the well plate manufacturer. It is further noted that imaging of well plates from below, as shown in this prior art system, is generally preferred for a number of reasons. First, there is generally better optical access from below, since the side walls do not need to be avoided. Second, it avoids having to image through the solution contained in the well. This is problematic, since the volume of liquid solution varies and thus the height of the upper surface of the liquid. Moreover, the upper surface of the liquid can move and inherently is not flat owing to meniscus effects.
With regard to focusing a camera to image the cells, a standard autofocus system may be adequate. However, for a container requiring many images, such as a well plate comprising 96, 384 or 1536 wells, it can be very time-consuming to refocus the camera for each well. This is particularly problematic if no stains or fluorescent tags are used to highlight the cells; the visual contrast between the cells and their surroundings can be insufficient for the optical feedback in the autofocus system to function efficiently. As an example, under these conditions it can take over an hour to image each well in a 96-well plate by refocusing the camera for every well, which is inconveniently slow for an automated system intended to handle many cell samples. Examples of such systems include the Nikon PFS (“perfect focus”™) system and the Olympus ZDS (“zero drift”™) system.
FIG. 3 schematically illustrates another prior art laser-based autofocus system of the type which is used by Molecular Devices Corporation in their automated microscope system sold under the trade name ImageXpress MICRO™. A laser beam 27 from a laser 26 is directed at a glancing angle towards the transparent base 24 of a sample container 21, and the reflections from both an upper surface 24a and a lower surface 24b of the base 24 are detected by a detector 28. As a result of the laser being directed at the base at a glancing angle, the reflected light from the upper surface 24a will take a path 29b which is parallel to, but offset from, a path 29a taken by the reflected light from the lower surface 24b. The position of each path can be used to provide an indication of the location of the respective surface, and the distance between the two paths can be used to determine the thickness of the base. While this technique may function adequately for a perfectly flat base having perfectly flat upper and lower surfaces, in reality this may not be the case. For example, the biological sample container may be bowed, which will cause the light beams 29a and 29b to be divergent and no longer allow accurate measurement of the base thickness.
FIGS. 4A and 4B schematically illustrate another prior art solution which is described in US20070009395A1 [3]. In this solution, a well plate is held in a specially designed vacuum bed and sucked down so its base is pressed against an optically flat surface, thus ensuring that all wells lie in the same plane and thereby obviating the need to focus on every well prior to imaging. Referring to FIG. 4A, the holder includes a vacuum bed having an optically flat planar surface 36 for receiving a lower surface 34 of a biological sample container 31 and a perimeter seal 32 surrounding the vacuum bed. The seal is dimensioned to receive the lower perimeter edge of a standard well plate 31. The holder includes an exhaust outlet for evacuating the space under the well plate 31 so that the well bases 34 are urged against the optical flat 36, thereby ensuring that the bases of all the wells are coplanar with each other as shown in FIG. 4B. This enables many or all of the samples in a container to be imaged sequentially without the need to refocus an imaging camera for every sample. Instead, the camera can be focused just once on one sample in one region of the container, and the focus retained for imaging the remainder of the container. This significantly speeds up the time needed for handling each container. However, this method is only as accurate as the manufacturing tolerances in the thickness of the material between the base of the well plate and the base of each well.